This invention is used in the laboratory determination of hemoglobin S, the abnormal hemoglobin which is responsible for Sickle Cell anemia. It is a solubility test. It is used along with electrophoresis or isoelectrofocusing procedures to make a definitive diagnosis for the presence of Hemoglobin S.
In U.S. Pat. No. 3,918,905 (Warren et al.), which is incorporated by reference herein, is disclosed a composition for determining the presence of sickling heterozygous or homozygous hemoglobin. That composition utilizes a two-phase composition comprising an aqueous phase containing a phosphate buffer system, a stable reducing agent system and a lysing agent. The aqueous phase is in combination with an organic phase, such as toluene, which is immiscible with the aqueous phase. By adding blood products to the claimed composition and performing the necessary steps of the analysis, one obtains a colorimetric determination of the genotype of the hemoglobin tested. For example, in that test, which produces various colored bands in the various immiscible phases, normal blood produces a middle band which is grey colored and the lower phase is red. A hemoglobin genotype of AS produces a middle band of dark red and a lower phase of pink. When the hemoglobin genotype is SS, the middle band is dark red and the lower phase is straw-colored or pale yellow.
While that method is capable of determining the hemoglobin genotype, it suffers from the deficiency of requiring an organic and toxic solvent such as toluene.
In the present invention, mineral oil has replaced the organic solvent such as toluene found in U.S. Pat. No. 3,91 8,905. Toluene, as the lighter immiscible phase, was the choice in 1975 because it had long been used as a lysing agent in hematology and as a preservative of biological fluids.
The aim of this invention is to eliminate the hazardous component toluene. Mineral oil is much safer. Mineral oil has a flash point of 177.degree. C. Toluene has a flash point of 4.degree. C. Mineral oil is not listed as hazardous by OSHA (Occupational Safety and Health Administration) or DOT (Department of Transportation). Also disposal problems are eliminated.
The results of the present invention are surprising and unexpected since the literature has reported that only toluene as the upper phase permits the accurate identification. In the American Journal of Medical Technology, Vol. 41, No. 9, September 1975, pages 317-321, Warren et al. reported on page 319, .paragraph.13 that toluene as the upper phase appeared to be the most effective organic solvent of the several that were tried.
Accordingly, a need exists for a method and composition of determining hemoglobin genotype which does not utilize organic solvents.